Protocol

OmniStain IMMUNO Protocol

September 21, 2026

Introduction

  • OmniStain IMMUNO (also known as INSIHGT) is a multiplexed 3D tissue immunostaining method.
  • Increases antibody penetration depth and uniformity by 20-30x.
  • The staining typically takes 4 days for whole adult mouse brains, or equivalently sized tissues (~1cm³-sized) and does not require specialized equipment or special tissue pre-treatment.
  • Fluorophore-conjugated primary antibodies are NOT required. To date, >360 commercially available primary antibodies have been validated to be compatible under specific conditions.
  • The use of methanol quenches fluorescent proteins.
  • For FFPE tissues, use OmniStain REZCUE to retrieve antigens first.
  • To achieve optimal nuclear stain penetration, refer to the OmniStain H&E + IMMUNO protocol.

Read the paper: Yau et al. Nature communications. 2024. 15: 10888.

Materials provided in the kit

  • 2x OmniStain buffer A
  • 1x OmniStain buffer B
  • 1x OmniStain buffer C

OmniStain IMMUNO Protocol

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** Incubate at 20-25°C unless otherwise specified.

** Use fresh solution for each repeat. Keep the specimen fully submerged at all times and agitate on an orbital shaker at 100 rpm, and place the tube horizontally to maximize fluid flow unless otherwise specified.

Tissue Pretreatment

  1. Place the tissue in a 50 mL conical tube.
  2. Dehydrate in 25 mL 50% v/v methanol in H₂O 2 x 30 min, then in 25 mL 100% methanol 3 x 30 min.
  3. Delipidate in 25 mL 2:1 v/v dichloromethane:methanol mix for 6-18 hours in a fume hood.
  4. Rehydrate and wash sequentially in 25 mL 100% methanol 3 x 30 min, 50% v/v methanol in H₂O 2 x 30 min, and PBSN 2 x 30 min.
  5. Pre-incubate in 2 mL 1x OmniStain buffer A at 37°C with the tube upright for 6-18 hours.
    – Prepare the 1x buffer A by mixing equal volumes of 2x OmniStain buffer A and PBSN.

    OmniStaining
    (values were illustrated for 1cm³ tissues or whole adult mouse brains and can be scaled)
  6. Discard the preincubation solution and add 1 mL 2x OmniStain buffer A.
  7. Add 10 μg of each primary antibody or lectin per 1 cm³ of tissue or per whole adult mouse brain as a starting amount. Directly conjugated primary antibodies and lectins may be included.
    – Reduce the amount only after validation for the individual probe and specimen type.
    – You can add multiple species-compatible primary antibodies for multiplexed 3D IHC.
  8. (Optional nuclear stain:) In a separate tube, mix 3 μL of 1 mM nuclear-dye stock with 3 μL 1x OmniStain buffer C; add the entire mixture to the staining solution.
    – If nuclear penetration is suboptimal, use the separate OmniStain H&E + IMMUNO protocol.
  9. Top up with PBSN or 2x OmniStain buffer A to reach a final 1x OmniStain buffer A concentration in the staining solution. To do so, let V (in mL) equal the combined volume added in Steps 7-8:
    – if V < 1 mL add (1 – V) mL PBSN.
    – if V > 1 mL, add (V – 1) mL 2x OmniStain buffer A.
  10. If necessary, add the minimum volume of 1x OmniStain buffer A with the same concentration of probes to fully submerge the tissue.
  11. Incubate and agitate the tube upright at 20-25°C for at least 4 days in the dark.
    – If penetration is suboptimal, extending the incubation time to up to 14 days may help.

    Post-primary Incubation
  12. Replace with 5 mL 1x OmniStain buffer B at 20-25°C for 6-18 hours, agitate the tube upright.
  13. Wash in 25 mL PBSN (3 x 2 hours), shake the tube horizontally.
    – If all primary antibodies and lectins are directly fluorophore-conjugated, proceed directly to Step 21.

    OmniStaining – Secondary
  14. Pre-incubate in 2 mL 1x OmniStain buffer A at 20-25°C for 6-18 hours while agitating the tube upright.
  15. Discard the preincubation solution and add 1 mL 2x OmniStain buffer A.
  16. Add secondary reagents based on the mass of each unconjugated primary antibody used in Step 7.
    – Add 1 μg of secondary IgG or Fab fragment per 1 μg of the corresponding primary antibody.
    – Add 0.4 μg of nanobody per 1 μg of the corresponding primary antibody.
  17. Top up with PBSN, 2x OmniStain buffer A or 1x OmniStain buffer A, using the dilution and coverage rules in Steps 9-10.
  18. Incubate and agitate the tube upright at 20-25°C for 12-18 hours in the dark.

    Post-secondary Incubation and Clearing
  19. Replace with 5 mL 1x OmniStain buffer B at 20-25°C for 6-18 hours, agitate the tube upright.
  20. Wash in 25 mL PBSN (3 x 2 hours), shake the tube horizontally.
  21. Dehydrate in 25 mL 50% v/v methanol in H₂O 2 x 30 min, then in 25 mL 100% methanol 3 x 30 min.
  22. Clear in 20 mL Omni CLEAR. Laboratory-prepared BABB consisting of benzyl alcohol:benzyl benzoate at 1:2 v/v, DBE or ECi may be used as alternatives. Replace with 20 mL fresh clearing solution after 3 h and continue for at least 6 additional hours, or until the tissue is optically clear.